Progression through mitosis presents a challenge to the maintenance of cellular transcriptional programs by forcing the eviction of RNA Polymerase II and of most transcriptional regulators from chromatin. Recent work has identified a number of transcription factors and histone post-translational modifications that remain associated with mitotic chromatin and may thus provide cells with a bookmark of their transcriptional programs and cellular identity. Here we will combine focused mechanistic experimentation with global analyses to study principles of mitotic bookmarking. We found that the essential erythroid transcription factor GATA1 is stably retained at a subset of its interphase binding sites during mitosis. Mitotic chromatin binding by GATA1 is necessary for the timely reactivation of select erythroid lineage specific genes following mitosis. In Aim 1 we will determine the mechanism and function of GATA1 binding to a subset of its interphase binding sites during mitosis. Computational analysis failed to provide clues as to what separates interphase only sites (I-sites) from sites at which GATA1 is retained (IM-sites). However, preliminary data indicate that I- and IM-binding characteristics are recapitulated when genomic GATA1 binding elements are ectopically inserted into the erythroid genome. This indicates that local sequence context determines I vs IM binding and provides a convenient assay to analyze the specificity of these elements. We will define the minimal sequence context necessary and sufficient for GATA1 retention on mitotic chromatin through truncations and point mutations. We will use genome editing to directly test in both gain- and loss-of-function studies the role of endogenous I- and IM-sites on gene reactivation following mitosis. Following the identification of cis-element(s) required for GATA1 IM characteristics, we will identify and functionally study the relevant trans acting factor(s) in GATA1 mediated bookmarking in erythroid cells. In Aim 2 in collaboration with the lab of Dr. Ben Garcia we will examine the dynamics of histone modifications on a global scale by quantitative mass spectrometry using highly purified mitotic erythroid cell populations. This will be the first characterization of global histone mark levels during mitosis. Select dynami and stable marks, as well as the enzymes placing them, will be studied by genome wide location analysis. We will then examine the impact of proteins that bind IM-site enriched histone marks in promoting GATA1 mitotic chromatin occupancy and in gene reactivation. These studies are expected to provide a better understanding of chromatin based and transcription factor based mechanisms of mitotic bookmarking in erythroid cells and might have implications for understanding the transcriptional stability as well as plasticity associated with cellular differentiation and lineage stability.